Quantitative studies and taste re-engineering experiments toward the decoding of the nonvolatile sensometabolome of Gouda cheese

The first comprehensive quantitative determination of 49 putative taste-active metabolites and mineral salts in 4- and 44-week-ripened Gouda cheese, respectively, has been performed; the ranking of these compounds in their sensory impact based on dose-over-threshold (DoT) factors, followed by the confirmation of their sensory relevance by taste reconstruction and omission experiments enabled the decoding of the nonvolatile sensometabolome of Gouda cheese. The bitterness of the cheese matured for 44 weeks was found to be induced by CaCl2 and MgCl2, as well as various bitter-tasting free amino acids, whereas bitter peptides were found to influence more the bitterness quality rather than the bitter intensity of the cheese. The DoT factors determined for the individual bitter peptides gave strong evidence that their sensory contribution is mainly due to the decapeptide YPFPGPIHNS and the nonapeptides YPFPGPIPN and YPFPGPIHN, assigned to the casein sequences beta-CN(60-69) and beta-CN(60-68), respectively, as well as the tetrapeptide LPQE released from alphas1-CN(11-14). Lactic acid and hydrogen phosphate were identified to play the key role for the sourness of Gouda cheese, whereas umami taste was found to be due to monosodium L-glutamate and sodium lactate. Moreover, saltiness was induced by sodium chloride and sodium phosphate and was demonstrated to be significantly enhanced by L-arginine.     

Sensomics mapping and identification of the key bitter metabolites in Gouda cheese

Application of a sensomics approach on the water-soluble extract of a matured Gouda cheese including gel permeation chromatography, ultrafiltration, solid phase extraction, preparative RP-HPLC, and HILIC combined with analytical sensory tools enabled the comprehensive mapping of bitter-tasting metabolites. LC-MS-TOF and LC-MS/MS, independent synthesis, and sensory analysis revealed the identification of a total of 16 bitter peptides formed by proteolysis of caseins. Eleven previously unreported bitter peptides were aligned to beta-casein, among which 6 peptides were released from the sequence beta-CN(57-69) of the N terminus of beta-casein and 2 peptides originated from the C-terminal sequence beta-CN(198-206). The other peptides were liberated from miscellaneous regions of beta-casein, namely, beta-CN(22-28), beta-CN(74-86), beta-CN(74-77), and beta-CN(135-138), respectively. Six peptides were found to originate from alpha(s1)-casein and were shown to have the sequences alpha(s1)-CN(11-14), alpha(s1)-CN(56-60), alpha(s1)-CN(70/71-74), alpha(s1)-CN(110/111-114), and alpha(s1)-CN(135-136). Sensory evaluation of the purified, synthesized peptides revealed that 12 of these peptides showed pronounced bitter taste with recognition thresholds between 0.05 and 6.0 mmol/L. Among these peptides, the decapeptide YPFPGPIHNS exhibited a caffeine-like bitter taste quality at the lowest threshold concentration of 0.05 mmol/L.     

Identification of bitterness-masking compounds from cheese

Bitterness-masking compounds were identified in a natural white mold cheese. The oily fraction of the cheese was extracted and further fractionated by using silica gel column chromatography. The four fractions obtained were characterized by thin-layer chromatography and nuclear magnetic resonance spectroscopy. The fatty acid-containing fraction was found to have the highest bitterness-masking activity against quinine hydrochloride. Bitterness-masking activity was quantitated using a method based on subjective equivalents. At 0.5 mM, the fatty acid mixture, which had a composition similar to that of cheese, suppressed the bitterness of 0.008% quinine hydrochloride to be equivalent to that of 0.0049-0.0060% and 0.5 mM oleic acid to that of 0.0032-0.0038% solution. The binding potential between oleic acid and the bitter compounds was estimated by isothermal titration calorimetry. These results suggest that oleic acid masked bitterness by forming a complex with the bitter compounds.     

Taste active compounds in a goat cheese water-soluble extract. 2. Determination of the relative impact of water-soluble extract components on its taste using omission tests

The aim of this work was to determine the relative impact of water-soluble compounds on the gustatory properties of a goat cheese water-soluble extract (WSE). Using a semisynthetic model mixture (MWSE) previously elaborated in physicochemical and gustatory accordance with the cheese WSE (see part 1, Engel et al. J. Agric. Food Chem. 2000, 48, 4252-4259), omission tests were performed. Among the main taste characteristics of the WSE (salty, sour, and bitter), saltiness was explained by an additive contribution of sodium, potassium, calcium, and magnesium cations, whereas sourness was mainly due to a synergistic effect involving sodium chloride, phosphates, and lactic acid and bitterness was found to result from calcium and magnesium chlorides, the impact of which was partially masked by sodium chloride. In contrast, amino acids, lactose, and peptides did not have any significant impact on WSE taste properties. To quantify the contribution of the taste active compounds to bitterness and saltiness, stepwise multiple linear regressions were performed. Those contributions were expressed as a percentage of the considered taste characteristic intensity in the WSE. The model obtained allowed up to 97.4% of the perceived saltiness to be described and approximately 85% of the bitterness.     

New bitter-masking compounds: hydroxylated benzoic acid amides of aromatic amines as structural analogues of homoeriodictyol

Starting from the known bitter-masking flavanones eriodictyol and homoeriodictyol from herba santa some structurally related hydroxybenzoic acid amides of benzylamines were synthesized and evaluated as masking agents toward bitterness of caffeine by sensory methods. The closest structural relatives of homoeriodictyol, the hydroxybenzoic acid vanillylamides 5-9, were the most active and were able to reduce the bitterness of a 500 mg L(-1) caffeine solution by about 30% at a concentration of 100 mg L(-1). 2,4-Dihydroxybenzoic acid vanillylamide 7 showed a clear dose-dependent activity as inhibitor of the bitter taste of caffein between 5 and 500 mg L(-1). Additionally, it was possible to reduce the bitterness of quinine and salicine but not of the bitter peptide N-l-leucyl-l-tryptophan. Combinations of homoeriodictyol and amide 7 showed no synergistic or antagonistic changes in activity. The results for model compound 7 suggested that the hitherto unknown masking mechanism is probably the same for flavanones and the new amides. In the future, the new amides may be alternatives for the expensive flavanones to create flavor solutions to mask bitterness of pharmaceuticals or foodstuffs.     

Evolution of the composition of a selected bitter Camembert cheese during ripening: release and migration of taste-active compounds.

The aim of this study was to add to the understanding of changes in taste that occur during the ripening of a bitter Camembert cheese by the evolution of its composition. Physicochemical analyses were performed on rind, under-rind, and center portions of a Camembert cheese selected for its intense bitterness. At each of the six steps of ripening studied organic acids, sugars, total nitrogen, soluble nitrogen, phosphotungstic acid soluble nitrogen, non-protein nitrogen, Na, K, Ca, Mg, Pi, Cl, and biogenic amines were quantified in each portion. Changes in cheese composition seemed to mainly result from the development of Penicillium camemberti on the cheese outer layer. Migration phenomena and the release of potentially taste-active compounds allowed for the evolution of saltiness, sourness, and bitterness throughout ripening to be better understood. Apart from taste-active compounds, the impact of the cheese matrix on its taste development is discussed.     

Antipeptide antibodies recognizing plasmin sensitive sites in bovine beta-casein sequence

To investigate plasmin activity in cheese, we produced antibodies to bovine beta-casein with controlled specificity, suitable as markers of the integrity of the major bonds involved in its initial breakdown. Sixteen rabbits were immunized with synthetic substitutes for six plasmin-sensitive peptides. Antisera raised to the peptides (f20-39), (f40-56), (f94-113), (f184-202), and (f193-209) recognized beta-casein in ACP-ELISA, Western-blot, and biosensor assays. Casein in vitro hydrolysis by plasmin or chymosin reduced the detection of these determinants in ACP-ELISA, in agreement with the enzymatic sensitivity of bonds included within the binding sites, or in their neighborhood. Antiserum to (f20-39) in particular allowed the specific detection of plasmin cleavage at the bond generating gamma1-CN. Antisera to C-terminus preferentially detected the cleavage by chymosin. Immunoassays using these antibodies would allow in situ monitoring of significant proteolysis events without bias originated in the secondary degradation of the released peptides.     

Structure determination and sensory analysis of bitter-tasting 4-vinylcatechol oligomers and their identification in roasted coffee by means of LC-MS/MS

Aimed at elucidating intense bitter-tasting molecules in coffee, various bean ingredients were thermally treated in model experiments and evaluated for their potential to produce bitter compounds. As caffeic acid was found to generate intense bitterness reminiscent of the bitter taste of a strongly roasted espresso-type coffee, the reaction products formed were screened for bitter compounds by means of taste dilution analysis, and the most bitter tastants were isolated and purified. LC-MS/MS as well as 1-D/2-D NMR experiments enabled the identification of 10 bitter compounds with rather low recognition threshold concentrations ranging between 23 and 178 micromol/L. These bitter compounds are the previously unreported 1,3-bis(3',4'-dihydroxyphenyl) butane, trans-1,3-bis(3',4'-dihydroxyphenyl)-1-butene, and eight multiply hydroxylated phenylindanes, among which five derivatives are reported for the first time. In addition, the occurrence of each of these bitter compounds in a coffee brew was verified by means of LC-MS/MS (ESI-) operating in the multiple reaction monitoring (MRM) mode. The structures of these bitter compounds show strong evidence that they are generated by oligomerization of 4-vinylcatechol released from caffeic acid moieties upon roasting.     

Effect of the high-oleic trait on roasted peanut flavor in backcross-derived breeding lines

The high-oleic trait of peanut (Arachis hypogaea L.) has been suggested to have a positive impact on the roasted peanut sensory attribute. A series of lines derived by backcrossing the high-oleic trait into several existing cultivars were compared with their parent cultivars at locations in Florida, Georgia, North Carolina, and Texas. Breeders grew their high-oleic lines and parents in three-replicate tests at one or two locations. The Florida high-oleic line F435-2-3-B-2-1-b4-B-B-3-b3-b3-1-B was grown at each location. The test included normal- and high-oleic variants of F435, GK 7, NC 7, NC 9, Sunrunner, Tamrun 96, and Tamspan 90. Sound-mature kernel samples were roasted, ground into paste, and evaluated by a sensory panel using a 14-point flavor intensity unit (fiu) scale. Background genotype had an effect (P < 0.01) on the heritable sensory attributes roasted peanut, sweet, and bitter. Oleate level had a positive effect on roasted peanut intensity, increasing it by 0.3 fiu averaged across all seven background genotypes. However, the magnitude of improvement varied across background genotypes. The high-oleic trait had no effect or increased the intensity of the roasted peanut attribute in each background genotype. The increase was greatest in Tamrun 96 (+0.6 fiu, P < 0.05) and Spanish genotypes Tamspan 90 (+0.4 fiu, P < 0.05) and F435 (+0.4 fiu, P < 0.10). A change of 0.5 fiu or more should be perceptible to consumers. Interaction between oleate level and background genotype was detected for sweet (P < 0.10) and bitter (P < 0.01) attributes. The trait had an increasing effect on the bitter attribute only in the background genotype of Tamspan 90 (+0.7 fiu, P < 0.01). There was a nonsignificant increase in bitterness in the other Spanish background genotype, F435. Changes in bitterness in runner- and Virginia-type backgrounds were close to zero. Incorporation of the high-oleic trait into peanut cultivars is likely to improve the intensity of the roasted peanut attribute, but it may also increase the bitter attribute in Spanish genotypes.     

苦杏仁脱苦去毒的研究

通过单项比较筛选出混合酸盐法为最佳方法,采用L₈(4×2⁴)正交试验及对其结果进行直观和方差分析,确定出苦杏仁脱苦的影响因素依次为是否去皮、换药液次数、浸泡液温度、浸泡液用量、浸泡液种类;影响苦杏仁去毒的因素依次为浸泡液温度、换药液次数、浸泡液用量、是否去皮、浸泡液种类;但各因素的影响间均无显著差异。筛选出的混合酸盐法减少了换药液次数(即废水量),缩短了脱苦时间,保持了杏仁的色香味。最后对苦杏仁脱苦后的废水成功地进行了处理。     

Structural analogues of homoeriodictyol as flavor modifiers. Part III: short chain gingerdione derivatives

In order to find new flavor modifiers, various short chain gingerdione derivatives were synthesized as structural analogues of the known bitter masker homoeriodictyol and evaluated by a sensory panel for masking and sweetness enhancing activities. 1-(4-Hydroxy-3-methoxyphenyl)hexa-3,5-dione ([2]-gingerdione) and the homologue 1-(4-hydroxy-3-methoxyphenyl)hepta-3,5-dione ([3]-gingerdione) at concentration ranges 50-500 mg kg (-1) showed the most promising masking activity of 20-30% against bitterness of a 500 mg kg (-1) aqueous caffeine solution. Additionally, both compounds were able to reduce the bitterness of a 5 mg kg (-1) quinine solution by about 20%; however, the bitter tastes of salicine, the model peptide H-Leu-Trp-OH, and KCl solutions were not reduced. Whereas for bitter masking activity a vanillyl moiety seems to be important, some of the tested isovanillyl isomers showed an interesting sweet enhancing effect without exhibiting a significant intrinsic sweetness. The isomer 1-(3-hydroxy-4-methoxyphenyl)hexa-3,5-dione ([2]-isogingerdione) at 100 mg kg (-1) caused a significant and synergistic increase of 27% of sweet taste of a 5% sucrose solution.     

Tastes and structures of bitter peptide, asparagine-alanine-leucine-proline-glutamate, and its synthetic analogues

Asn-Ala-Leu-Pro-Glu (NALPE) is a strong bitter peptide with a minimum response threshold (MRT) of 0.074 mM. To elucidate the relationship of spatial structure and bitterness on peptides, NALPE and its analogues, NALPW, NALPS, NALPL, NALPP, NALPD, and NALPR, were synthesized and sensorially evaluated. Structural analysis using computer simulation for each peptide revealed that the presence of a polar group and hydrophobic bitter amino acids, the composition of hydrophobic regions, the spatial orientation of the polar group and hydrophobic regions, and the proximity between polar groups and hydrophobic regions faced within the same plane space may be the major determinants for the taste type and intensity of peptide bitterness.     

Immunochemical evaluation of bovine beta-casein and its 1-28 phosphopeptide in cheese during ripening

Polyclonal antibodies raised against the plasmin-released 1-28 phosphopeptide from bovine beta-casein [i.e., beta-CN(f1-28)4P] specifically recognized the tryptic beta-casein 1-25 and 2-25 peptides, whatever the degree of phosphorylation, but were unresponsive to the shortened beta-casein 16-22 phosphopeptide. These antibodies were able to recognize the parent bovine beta-casein as well as the homologous water buffalo protein, but they could not detect the homologous counterparts from ovine and caprine milks. Such antibodies were used in competitive enzyme-linked immunosorbent assays to monitor the plasmin-mediated release of the 1-28 phosphopeptide from beta-casein and to evaluate the residual native beta-casein in bovine cheese sampled during ripening. Applications of these polyclonal antibodies are suggested mainly for estimating the age of hard cheeses and, possibly, for tracing the presence of bovine casein in fresh ovine and caprine cheeses.     

Flavor enhancement of reduced fat cheddar cheese using an integrated culturing system

Mild cheese flavor in reduced fat Cheddar cheese was enhanced by using an integrated starter culture system. Three cultures, Lactococcus lactis subsp. cremoris SK11, L. lactis subsp. lactis biovar. diacetylactis JVI, and Lactobacillus casei 7A, were carefully selected to obtain a nonbitter, mildly acid, buttery flavored cheese. Cheeses were produced from all possible combinations of these cultures with the constraint that L. lactis subsp. cremoris SK11 was used as the primary acid-producing culture. Cheeses made with SK11 were compared to cheeses produced using an L. lactis subsp. cremoris commercial starter culture. Cheeses were ripened for 150 days and periodically sampled for chemical, microbiological, and sensory analysis. Cheeses produced with L. lactis subsp. cremorisSK11 had substantially lower bitterness intensity than the cheeses produced with commercial starter culture. L. lactis subsp. lactis biovar. diacetylactis JVI significantly increased diacetylacetoin and acetate concentrations. Sensory results indicate that these cheeses had increased buttery (diacetyl) flavor.     

Flavor-active compounds potentially implicated in cooked cauliflower acceptance

The aim of the present study was to determine the flavor-active compounds responsible for the "sulfur" and "bitter" flavors of cooked cauliflower potentially implicated in cauliflower rejection by consumers. Eleven varieties of cauliflower were cooked and assessed by a trained sensory panel for flavor profile determination. Among the 13 attributes, the varieties differed mainly according to their "cauliflower odor note" and their "bitterness". Various glucosinolates were quantified by HPLC and correlated with bitterness intensity. The results showed that neoglucobrassicin and sinigrin were responsible for the bitterness of cooked cauliflower. Application of Dynamic Headspace GC-Olfactometry and DH-GC-MS showed that allyl isothiocyanate (AITC), dimethyl trisulfide (DMTS), dimethyl sulfide (DMS), and methanethiol (MT) were the key odorants of cooked cauliflower "sulfur" odors. Moreover, these volatile compounds corresponded to the main compositional differences observed between varieties. Finally, AITC, DMTS, DMS, MT, sinigrin, and neoglucobrassicin were shown to be potential physicochemical determinants of cooked cauliflower acceptance.     

Proteolytic activities of suparen and rennilase on buffalo, cow, and goat whole casein and beta-casein.

The proteolytic specificity and activity of Mucor miehei protease (Rennilase) and Endothia parasitica protease (Suparen) on buffalo, cow, and goat whole casein and beta-casein (CN) were studied by analyzing the degradation products. The results suggest that Rennilase hydrolyzes casein of the three species in a manner similar to that of chymosin, resulting in the formation of alpha(s1)-I and beta-I, -II, -III as initial degradation fragments of alpha(s1)- and beta-CN. alpha(s1)-I was also the initial breakdown product of alpha(s1)-CN by Suparen. Contrary to Rennilase, Suparen showed a higher affinity toward beta-CN and hydrolyzes beta-CN, giving rise to degradation products characterized by mobility lower than that of beta-CN. Increasing NaCl concentration (>3%) reduced the proteolysis of beta-CN of the three species by Rennilase but not by Suparen. The hydrolysis of alpha(s1)-CN and alpha(s1)-I by the two enzymes was enhanced in the presence of NaCl.     

Structure determination of 3-O-caffeoyl-epi-gamma-quinide, an orphan bitter lactone in roasted coffee

Recent investigations on the bitterness of coffee as well as 5- O-caffeoyl quinic acid roasting mixtures indicated the existence of another, yet unknown, bitter lactone besides the previously identified bitter compounds 5- O-caffeoyl- muco-gamma-quinide, 3- O-caffeoyl-gamma-quinide, 4- O-caffeoyl- muco-gamma-quinide, 5- O-caffeoyl- epi-delta-quinide, and 4- O-caffeoyl-gamma-quinide. In the present study, this orphan bitter lactone was isolated from the reaction products generated by dry heating of 5- O-caffeoylquinic acid model, and its structure was determined as the previously unreported 3- O-caffeoyl- epi-gamma-quinide by means of liquid chromatography-mass spectrometry (LC-MS) and one-/two-dimensional NMR experiments. The occurrence of this bitter lactone, exhibiting a low bitter recognition threshold of 58 micromol/L, in coffee beverages could be confirmed by LC-MS/MS (negative electrospray ionization) operating in the multiple reaction monitoring mode.     

Quantitative structure-activity relationship study of bitter peptides

A database consisting of 224 di- to tetradecapeptides and five amino acids was compiled to study quantitative structure-activity relationships of bitter peptides. Partial least-squares regression-1 analysis was conducted using the amino acid three z-scores and/or three parameters (total hydrophobicity, residue number, and log mass values) as X-variables and bitterness values (log 1/T where T is the bitterness threshold) as Y-variables. Using the three parameters only, significant models (p < 0.001) were obtained describing the entire data set as well as data subsets, except that comprised only of octa- to tetradecapeptides. For data sets comprising different peptide lengths, the models were improved by including the three z-scores at the N-terminal and C-terminal positions. Correlation coefficients for bitterness prediction of 48 dipeptides and 12 pentapeptides were 0.75 (RMSEP = 0.53) and 0.90 (RMSEP = 0.48), respectively. Bulky hydrophobic amino acids at the C terminus and bulky basic amino acids at the N terminus were highly correlated to bitterness.     

Evolution of the taste of a bitter Camembert cheese during ripening: characterization of a matrix effect.

The objective of this study was to characterize the effect of ripening on the taste of a typically bitter Camembert cheese. The first step was to select a typically bitter cheese among several products obtained by different processes supposed to enhance this taste defect. Second, the evolution of cheese taste during ripening was characterized from a sensory point of view. Finally, the relative impact of fat, proteins, and water-soluble molecules on cheese taste was determined by using omission tests performed on a reconstituted cheese. These omission tests showed that cheese taste resulted mainly from the gustatory properties of water-soluble molecules but was modulated by a matrix effect due to fat, proteins, and cheese structure. The evolution of this matrix effect during ripening was discussed for each taste characteristic.